title: Single-molecule analysis of the rotation of F₁-ATPase under high hydrostatic pressure.
abstract: F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F1 actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F1 has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F1(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å(3) and +88 Å(3) for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F1 and pressure-induced protein unfolding.
authors.affiliation: Laboratory for Cell Dynamics Observation, Quantitative Biology Center, Riken, Furuedai, Suita, Osaka, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Okuno
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Nishiyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006874:Hydrostatic Pressure; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012399:Rotation
chemical_list: D001426:Bacterial Proteins; D006180:Proton-Translocating ATPases